Process for the production of proteins and the production of arrays of proteins

ABSTRACT

Described is a novel process for the production of at least one protein of interest by secretion of the protein of interest from a pro- or eukaryotic host cell in a compartment system, which host cell is stably expressing a secretion system and capable of heterologous secretion of the protein of interest and which compartment system has at least a first and a second compartment and wherein the host cell is located in the first compartment and wherein the first and second compartment are separated from each other by a barrier, wherein the barrier is permeable for the secreted protein of interest, but not permeable for the host cell.

This application is a 371 of PCT/EP01/15008, filed Dec. 19, 2001, whichclaims the benefit of U.S. patent application Ser. No. 60/307,166, filedJul. 24, 2001 and U.S. patent application Ser. No. 60/256,456, filedDec. 20, 2000.

TECHNICAL FIELD

The present invention relates to a process for the production andpurification of proteins of interest by secretion of the protein ofinterest from a host cell in a compartment system, to a process for theproduction of protein arrays, to a suitable compartment system and tohost cells and plasmids suitable for the production and secretion of theproteins of interest.

PRIOR ART

The current technology on the creation of protein and proteome arrays isbased upon cloning of open reading frames (ORFs) encoding certainproteins of interest into expression vectors and their expression bycorresponding bacterial cells carrying the respective vectors. Followinga gentle lysis of the bacteria on filters, the bacterial debris iswashed off, and the filter-bound proteins are utilized for bindingstudies. [Büssow et al. (1998) Nucl. Acids Res. 26:5007–5008; Walter etal. (2000) Curr. Op. Microbiol. 3:298–302]. The drawbacks of this methodare (i) that due to the fact that all proteins produced by thesebacteria are present on the filter, there is a high background ofbacterial proteins which can interfere with specific binding studies,and (ii) that the proteins of interest may be denatured during bacteriallysis. The problem of a high background level can be solved bypurification of the overexpressed proteins, however, this is very labourintensive.

Surprisingly it was found now, that the drawbacks of the currenttechnology can be avoided by a novel process which make use of asecretion system which ensures the secretion of a protein of interestfrom a host cell in a compartment system which allows the separation ofthe protein of interest form its synthesizing host cell and itscomponents and allows the preservation of the protein of interest in anundenatured form.

DESCRIPTION OF THE INVENTION

A first subject of the present invention is therefore a process for theproduction of at least one protein of interest by secretion of theprotein of interest by a host cell into a compartment system, which hostcell is stably expressing a secretion system and capable of heterologoussecretion of the protein of interest and which compartment system has atleast a first and a second compartment and wherein the host cell islocated in the first compartment and wherein the first and secondcompartment are separated from each other by a barrier, wherein thebarrier is permeable for the secreted protein of interest, but notpermeable for the host cell. Optionally the protein of interest isretained in the second compartment and may be collected from the secondcompartment. This novel process ensures the secretion of the proteins ofinterest into a compartment system which allows their separation fromthe synthesizing host cell and all of its components and preserves theproteins in a non-denatured form as no lysis of the host cell isnecessary. As an option the novel process allows the retention of thesecreted protein on a carrier and/or purification by use of an affinitytag, and as further option allows their detection by use of anepitope-tag and as another option allows the removal of secretion signaland tag sequences which might affect the function of the protein ofinterest by introduction of suitable protease cleavage sites.

Another subject of the present invention is a process for the productionof a protein array. In connection with the invention the term proteinarray refers to an ordered arrangement of individual proteins ofinterest, in particular to an ordered arrangement of individualproteins, allowing the parallel analysis of the proteins. Preferentiallythe organisational principle is partitioning, using either tubes,cavities or discrete patches or spots on planar surfaces [Walter et al.(2000) Curr. Op. Microbiol. 3:298–302]. The invention allows thesecretion of a protein of interest from a host cell which grows in onecomponent of a compartment system into another component of the samecompartment system. The host cells are expressing a secretion system,which enables the host cells to secrete heterologous proteins ofinterest. The compartment system has at least a first and a secondcompartment, wherein the different host cells are arranged in an arrayform in said first compartment and wherein the first and secondcompartment are separated from each other by a barrier, wherein thebarrier is permeable for the secreted proteins of interest, but notpermeable for the host cell and its other components and wherein thesecreted proteins of interest are received in the second compartment inthe form of an array.

A still further subject of the invention is a protein array obtainableby a process according to the present invention. Such array will onlycontain proteins secreted by the host cell constructs.

Protein of interest in connection with the present invention refers toany protein of an organism of interest, which may be of viral,prokaryotic or eukaryotic nature, which can be secreted by a suitablesecretion system.

Secretion of the protein of interest from a host cell in connection withthe invention refers to the transport of the protein of interest formthe producing host cell into the extracellular space.

Secreted protein in connection with the invention refers to any proteinof interest which can be secreted by a suitable secretion system, inparticular to a protein of interest secreted as a fusion protein withthe C-terminal signal sequence of the E. coli α-hemolysin protein HlyAor with the α-factor signal sequence from Saccharomyces cerevisiae.

Host cell refers to any cell which is able to secrete heterologousproteins of interest in particular to apathogenic Gram-negativebacteria, preferentially to apathogenic E.coli, in particular E.coliK-12, or Pichia pastodis yeast cells.

Secretion system in connection with the invention refers to anyprokaryotic or eukaryotic secretion system which allows the secretion ofheterologous proteins of interest, in particular to either a secretionsystem of Gram negative bacteria [Sandkvist and Bagdasarian (1996) Curr.Op. Biotechnol. 7:505–511], preferentially to the type I secretionsystem of Gram negative bacteria, preferentially to the E. coliα-hemolysin secretion system, as described in the following publications[Dietrich et al. (1998) Nature Biotechnol. 16:181–185; Gentschev et al.(1994) Behring Inst. Mitt. 95:57–66; Gentschev et al. (1996) Mol. Gen.Genet. 252:266–274; Gentschev et al. (1996) Gene. 179:133–140; Gentschevet al. (1997) Behring Inst. Mitt. 98:103–113; Gentschev et al. (1998)Infect. Immun. 66:2060–2064; Mollenkopf et al. (1996) BioTechniques.21:854–860; Spreng and Gentschev (1998) FEMS Microbiol. Lett.165:187–192; Spreng et al. (1999) Mol. Microbiol. 31:1589–1601], inparticular to a modified version of the E. coli α-hemolysin secretionsystem, or to a secretion system of Gram positive bacteria [Braun et al.(1999) Curr. Op. Biotechnol. 10:376–381; Ferrari et al. (1993) in A. L.Sonenshein et al. (eds). Bacillus spp. and other Gram-positive bacteria.Am. Soc. Microbiol, Washington, D.C., p. 917–937.; Freudl (1992) J.Biotechnol. 23:231–240; Pozidis et al. (2001) Biotechnol. Bioeng.72:611–619; Van Wely et al. (2001) FEMS Microbiol. Rev. 25:437–454],preferentially Bacillus spp., Staphlyococcus spp., Streptomyces spp., orto yeast secretion systems, preferentially those mediated by theSaccharomyces cerevisiae α-factor or the Pichia pastotris acidphosphatase (PHO1) signal sequence [Cereghino and Cregg (2000) FEMSMicrobiol. Rev. 24:45–66; these yeast secretion systems are alsocommercially available from Invitrogen Corporation].

It is preferred that secretion system in connection with the inventionrefers to any prokaryotic or eukaryotic secretion system which allowsthe secretion of heterologous proteins of interest, in particular to theα-hemolysin secretion system, preferentially to the E.coli α-hemolysinsecretion system, in particular to a modified version of the E. coliα-hemolysin secretion system [Dietrich et al. (1998) Nature Biotechnol.16:181–185; Gentschev et al. (1994) Behring Inst. Mitt. 95:57–66;Gentschev et al. (1996) Mol. Gen. Genet. 252:266–274; Gentschev et al.(1996) Gene. 179:133–140; Gentschev et al. (1997) Behring Inst. Mitt.98:103–113; Gentschev et al. (1998) Infect. Immun. 66:2060–2064;Mollenkopf et al. (1996) BioTechniques. 21:854–860; Spreng and Gentschev(1998) FEMS Microbiol. Lett. 165:187–192; Spreng et al. (1999) Moi.Microbiol. 31:1589–1601], or to a modified version of the Pichiapastoris secretion system commercially available fromlnvitrogen.

In connection with the invention secretion of a heterologous proteinrefers to the secretion of a protein by a host cell which may notnecessarily be part of the natural proteome of the host cell but issynthesized by the host cell due to the fact that the corresponding genewas introduced into the host cell genome. In connection with theinvention secretion of the respective heterologous protein by the hostcell refers to its transport from the host cell into the extracellularspace by a secretion machinery, in particular as a fusion between theheterologous protein of interest and the C-terminal HlyA signal sequenceby the α-hemolysin secretion system or the Saccharomyces cerevisiaeα-factor or the Pichia pastoris acid phosphatase (PHO1) signal sequence.

Host cell stably expressing a secretion system and capable ofheterologous secretion of the protein in connection with the inventionrefers to a host cell which is able to express a secretion system thatis capable of inducing the secretion of a protein, the correspondinggene of which was introduced into its genome. In particular, capable ofheterologous secretion of the protein refers to the capability tosecrete a heterologous protein. The stable expression of the secretionsystem can be acchieved by assuring that the secretion system ischromosomally encoded, which can be the case either by nature or due togenetic engineering. It is also preferred that for stable expression ofthe secretion system components of the secretion system are cloned on amobile vector, e.g. plasmid, also encoding the heterologous proteins ofinterest to be secreted. It is also preferred that for stable expressionof the secretion system the secretion system and the heterologousprotein of interest are cloned on separate, compatible mobile vector,e.g. plasmids.

In the following, a prokaryotic secretion system, based on the E. coliα-hemolysin secretion system and a eukaryotic secretion system, based ona Pichia pastoris secretion system, will be described:

Prokaryotic Secretion System

Prokaryotic secretion system refers to the application of a prokaryoticcell as a host cell for the secretion of a heterologous protein ofinterest, in particular to either apathogenic Gram-negative bacteria,preferentially to apathogenic E. coli, in particular E. coli K-12 or Bstrains, or apathogenic Gram-positive bacteria, preferentially Bacillusspp., Staphlyococcus spp., Streptomyces spp.

In connection with the invention, secretion of the respectiveheterologous protein by a prokaryotic host cell refers to its transportfrom the prokaryotic host cell into the extracellular space by asecretion machinery.

Prokaryotic E. coli α-hemolysin Secretion System

The α-hemolysin is a pathogenicity factor which is frequently producedby extraintestinal E. coli pathogens, predominantly by uropathogenic E.coli (UPEC). It is a membrane-damaging, pore-forming extracellularcytotoxin belonging to the Rtx (repeats in toxin) family of proteintoxins. The HlyA protein lysis eukaryotic cells, including erythrocytes,because of which It was termed “hemolysin”, but also other cells, suchas granulocytes and epithelial cells. Rtx cytotoxins are produced by avariety of Gramnegative bacteria and are characterized by a C-terminalcalcium-binding region with a variable number of glycine-rich repeatunits consisting of nine amino acids. Calcium-binding of the releasedRtx toxins in the extracellular space is essential for their cytotoxicactivities. They are transported across both the inner and outermembranes of Gram-negative bacteria by the sec-independent type Isecretion pathway. While in the majority of hemolytic E. coli pathogens,the hly determinants are chromosomally encoded, in about 5% of them thehly genes are located on plasmids. [Ludwig and Goebel (1991) In:Sourcebook of bacterial protein toxins, Eds. Alouf, J. E., Freer, J. H.,Academic Press, pp. 117–125; Mühldorfer and Hacker (1994) Microb.Pathogen. 16:171–181].

The synthesis and secretion of the E. coli α-hemolysin is encoded by anoperon consisting of four genes, designated hlyC, hlyA, hlyB and hlyD[Ludwig and Goebel (1991) In: Sourcebook of bacterial protein toxins,Eds. Alouf, J. E., Freer, J. H., Academic Press, pp. 117–125]. WhilehlyA codes for the structural HlyA cytolytic protein, the co-synthesizedHlyC protein is required for HlyA activation within the bacterialcytosol by posttranslationally converting the nontoxic prohemolysin(proHlyA) into the toxic α-hemolysin (HlyA) by fatty acylation of twointernal lysine residues [Ludwig and Goebel (1991) In: Sourcebook ofbacterial protein toxins, Eds. Alouf, J. E., Freer, J. H., AcademicPress, pp. 117–125; Stanley et al. (1998) Microbiol. Mol. Biol. Rev.62:309–333; Stanley et al. (1999) Mol. Microbiol. 34:887–901; Trent etal. (1998) Biochemistry. 37:4644–4652; Trent et al. (1999) Biochemistry.38:9541–9548]. HlyB and HlyD are required for the transport of HlyAacross the bacterial membranes [Ludwig and Goebel (1991) In: Sourcebookof bacterial protein toxins, Eds. Alouf, J. E., Freer, J. H., AcademicPress, pp. 117–125]. Moreover, the outer membrane protein TolC, which ischromosomally encoded and not part of the α-hemolysln operon, is part ofthe α-hemolysin secretion apparatus [Ludwig and Goebel (1991) In:Sourcebook of bacterial protein toxins, Eds. Alouf, J. E., Freer, J. H.,Academic Press, pp. 117–125; Schlör et al. (1997) Mol. Gen. Genet.256:306–319]. The signal sequence within the C-terminal 60 amino acidsof HlyA, which contains a helix(α1)-linker-helix(α2) motif, issufficient for being recognized by the secretion apparatus [Koronakis etal. (1989) EMBO J. 8:595–605; Jarchau et al., (1994) Mol. Gen. Genet.245:53–60; Hul et al. (2000) J. Biol. Chem. 275:2713–2720], consistingof the membrane proteins HlyB, HlyD and TolC. The C-terminal peptide canalso be secreted by itself [Jarchau et al., (1994) Mol. Gen. Genet.245:53–60; Hui et al. (2000) J. Biol. Chem. 275:2713–2720]. It hasrecently been demonstrated that only the α1-amphiphilic helix and thelinker but not the second helix are essential for HlyA transport [Hui etal. (2000) J. Biol. Chem. 275:2713–2720]. HlyB, an inner membranetraffic ATPase, which energizes the transport of HlyA, recognizes theHlyA C-terminal signal sequence with its cytoplasmic domains, initiatesHlyA translocation and forms a transmembrane channel in the innermembrane through which HlyA is translocated. HlyD was suggested to serveas a linker between the inner and outer bacterial membrane. It isinserted in the inner membrane, but most of it is localized in theperiplasm where It was suggested to interact with both HlyB of the innermembrane and TolC of the outer membrane. By interacting with periplasmicHlyB loops, HlyD and HlyB form a channel through the periplasm. As HlyDalso has a TolC-homologous part, it is assumed to participate with TolCin the formation of a transmembrane channel in the outer membranethrough which HlyA is secreted. Consequently, HlyD is required forpulling HlyA through the bacterial membranes and for its release intothe extracellular space. In case of the plasmid-encoded α-hemolysin, itssynthesis and secretion in E. coli is enhanced by HlyR, a long distanceactivator which is encoded by the hlyR gene located at some distanceupstream of the hlyC gene [Vogel et al., (1988) Mol. Gen. Genet.212:76–84]. In contrast, a hlyR homologous activator gene has not beenfound in chromosomal hly determinants. [Ludwig and Goebel (1991) In:Sourcebook of bacterial protein toxins, Eds. Alouf, J. E., Freer, J. H.,Academic Press, pp.117–125].

It has previously been demonstrated that the hemolysin secretionapparatus works in a great variety of Gram-negative bacteria [Spreng etal. (1998) FEMS Microbiol. Lett. 165:187–192] and that it can beutilized for the secretion of heterologous proteins of different origin(e.g. bacterial, viral, protozoan) [Dietrich et al. (1998) NatureBiotechnol. 16:181–185; Gentschev et al. (1994) Behring Inst. Miff.95:57–66; Gentschev et al. (1996) Mol. Gen. Genet. 252:266–274;Gentschev et al. (1996) Gene. 179:133–140; Gentschev et al. (1997)Behring Inst. Mitt. 98:103–113; Gentschev et al. (1998) Infect. Immun.66:2060–2064; Mollenkopf et al. (1996) BioTechniques. 21:854–860; Sprengand Gentschev (1998) FEMS Microbiol. Lett. 165:187–192; Spreng et al.(1999) Mol. Microbiol. 31:1589–1601]. This was acchieved by generatinggene fusions between heterologous genes and the C-terminal signalsequence of hlyA, coding for secretion-competent hybrid proteins whichcan be secreted when HyB, HlyD and TolC are synthesized by therespective bacteria. Thus, it was shown to be of value as an antigendelivery system for the presentation of secreted antigens byGram-negative bacterial vaccine carriers. However, it became obviousthat there are great differences in the yields of heterologous proteinsecretion obtained with different proteins. Thus, the efficacy ofheterologous protein secretion via the hemolysin secretion system isdependend on the nature of the heterologous protein. E. g. proteinswhich have an N-terminal signal sequence and are usually secreted by thesec-dependent secretion pathway are only inefficiently transported bythe hemolysin secretion system [Gentschev et al. (1997) Behring Inst.Mitt. 98:103–113]. However, this problem can be circumvented byoptionally using a host cell harbouring a secA mutation [Gentschev etal. (1997) Behring Inst. Mitt. 98:103–113], which is a preferrred aspectin connection with the invention. Moreover, it has been demonstratedthat the efficacy of heterologous protein secretion via the hemolysinsecretion system correlates with the size of the heterologous gene infront of the HlyA_(s) signal [Spreng and Gentschev (1998) FEMSMicrobiol. Lett. 165:187–192]. As described below in greater detail,this problem is also faced in the invention by optionally binding theproteins of interest via an affinity tag onto a carrier with a suitablebinding partner until saturation will be achieved.

Eukaryotic Secretion System

Eukaryotic secretion system refers to the application of a eukaryoticcell as a host cell for the secretion of a heterologous protein ofinterest, in particular to apathogenic yeasts, e.g. Saccharomycescerevlsieae or Pichia pastors. In connection with the invention,secretion of the respective heterologous protein by a eukaryotic hostcell refers to its transport from the eukaryotic host cell into theextracellular space by a secretion machinery.

The yeast Pichia pastoris can relatively easy be geneticallymanipulated. As it is a eukaryotic organism, it is capable of manyposttranslavional modifications which occur also in higher eukaryotes,such as proteolytic processing, protein folding, disulfide bonding andglycosylations. Thus, many eukaryotic proteins which when expressed inbacteria are stored as inactive molecules in inclusion bodies, can beproduced by yeast cells as active molecules with all the necessaryposttranslational modifications. Compared with higher eukaryotic cells,yeast cells offer the advantage that they grow faster and that theirfermentation is easier and cheaper and that their yield of recombinantheterologous protein is higher [Cereghino and Cregg (1999) Curr. OpinionBiotechnol. 10:422–427; Dominguez et al. (1998) Int. Microbiol.1:131–142].

Pichia pastoris is a methylothrophic yeast, i.e. it is able tometabolize methanol. The first step in the methanol metabolism involvesthe oxidation of methanol to formaldehyde. This process is catalyzed bythe enzyme “Alkohol-Oxidase” (AOX). In order to prevent the toxic effectof hydrogen peroxide (a product of the enzyme reaction) for the yeastcell, the reaction takes place in a specialized organell, the so-calledperoxisome. The expression of the AOX1-gene is tightly and is induced byhigh amounts of methanol.

The P. pastoris expression and secretion systems is commerciallyavailable from invitrogen. These systems utilize the AOX-promoter forthe expression of heterologous genes. Culturing P. pastoris cells in afermenter in the presence of methanol provides yields of recombinantproteins up to 30% of the soluble total protein content of the culture.

P. pastoris can produce heterologous proteins either intracellulary orit can secrete those proteins into the extracellular medium. A shuttleplasmid vector, capable of replicating in E. coli and integrating intothe P. pastoris genome is provided, which can be used for the expressionand secretion of heterologous proteins, as it contains a sequenceencoding the α-factor-signal sequence from Saccharomyces cerevisiae,which guarantees the efficient secretion of heterologous proteins. As P.pastoris secretes only very little endogenous proteins and its growthmedium doesn't require proteins, the secreted heterologous protein formsthe main secretion product of a P. pastoris culture [Bretthauer undCastellino (1999) Biotechnol. Appi. Biochem. 30:193–200; Buckholz andGleeson (1991) Bio/Technology 9:1067–1072; Cereghino and Cregg (1999)Curr. Opinion Biotechnol. 10:422–427; Cereghino and Cregg (2000) FEMSMicrobiol. Rev. 24:45–66; Cregg et al. (2000) Mol. Biotechnol. 16:23–52;Dominguez et a. (1998) Int. Microbiol. 1:131–142; Gleeson et al. (1998)in: Methods in Mol. Biol. 103: Pichia Protocols:81–94; Stratton et al.(1998) In: Methods in Mol. Biol. 103: Pichia Protocols:107–120].

The P. pastoris secretion system can be used in connection with thepresent invention. Therefore, in analogy to the above described E. colihemolysin secretion system, plasmids derived from the above mentionedPichia pastoris vectors can be constructed, harbouring various genes ofinterest and optional various protease cleavage sites and tag sequences.

The compartment system according to the invention has at least a firstand a second compartment separated by a barrier. Preferably compartmentsystem refers to a system consisting of two locally partitioningsubsets, the first of which harbours the growing host cell either inliquid growth medium or on a planar surface, and the second of which isseparated from the first subset by a barrier which allows the flow ofproteins produced and secreted by the host cell from the first into thesecond subset but prevents host cell migration.

Barrier refers to a means of partitioning two components of acompartment system selectively preventing the flow-through of certainobjects from the first into the second component in particular, barrierrefers to a means of partitioning two compartments of a compartmentsystem, allowing the flow-through of proteins but retains other hostcell debris, in particular It refers to a membrane, preferably to afilter. In a preferred embodiment of this invention, a barrierpartitioning two compartments of a compartment system is part of thefirst compartment.

Examples for suitable compartmental systems are (i) a double filtersystem on a bacterial growth agar plate with bacterial colonies on thetop and secreted proteins on the bottom filter, as shown in FIG. 1 andas described in principle for the detection of Shiga-liketoxin-producing E. coli in fecal samples [Hull et at. (1993) J. Clin.Microbiol. 31:1167–1172], (ii) a system consisting of differentfilter-separated compartments containing liquid host cell cultures inone compartment and secreted protein solutions in the other compartment.Those two compartments are separated by a membrane that prevents hostcell migration but allows diffusion of proteins along theirconcentration gradient. On a small scale, e. g. this can be realized inform of a system consisting of stacked microwells which are separated bya membrane that prevents bacterial migration but allows diffusion ofproteins along their concentration gradient, as illustrated in FIG. 2.In the latter case, the top wells may contain host cell cultures whichsecrete fusions between the protein of interest and a suitable signalsequence (e. g. E. coli HlyA_(s)- or the yeast α-factor) as describedabove. While the host cells are forced by the filter to remain in thetop well, the secreted protein solutions diffuse into the bottom wellsand can be harvested as a protein solution. In case of the double filtertechnology, the top filter has to have a pore diameter that preventsbacterial migration from the top to the lower filter, but allows flow ofthe secreted fusion proteins to the lower filter. Preferably it shouldhave a low protein binding capacity. In contrast, the lower filter hasto have an optimal protein binding capacity, either by nature or as aconsequence of being coated at certain spots with a component (e. g.single stranded nucleic acids) which is an optimal binding partner for acomponent (e. g. C-terminal domain of A1 protein, [Cartegni et al.(1996) J. Mol. Biol. 259:337–348] of the protein fusion to be secretedby the bacteria growing on the top filter. Both filters must allow theflow-through of nutrients and growth factors from the bacterial growthagar plate to the bacterial colonies growing on the top filter. In caseof the technology using different, filter-separated compartmentscontaining liquid bacterial cultures in one compartment and secretedprotein solutions in the other compartment (e. g. stacked microwellsseparated by a filter), the separating filter has to have a porediameter that prevents bacterial migration but allows flow of thesecreted fusion proteins.

Prokaryotic System: Construction of a Gram Negative Bacterial Strain forHeterologous Protein Secretion Based in the E coli α-hemolysin SecretionSystem

In connection with the invention, the hlyB and hlyD genes are introducedinto the Gram-negative bacterial genome, harbouring a chromosomallyencoded tolC gene, in order to enable the bacteria to express thecomplete α-hemolysin secretion apparatus. In connection with theinvention, proteins of interest are made competent for secretion via theα-hemolysin secretion system by the creation of gene fusions between therespective heterologous genes of interest and the C-terminal signalsequence of HlyA (hlyA_(s)). The gene fusions are cloned on suitablemobile vectors, such as plasmids or cosmids. Consecutively, they areintroduced into a Gram negative bacterial strain which expresses thecomplete α-hemolysin secretion apparatus, consisting of HlyB, HlyD andTolC. The stable expression of the secretion system can be acchieved byinsertion of the hlyB and hlyD genes into the bacterial chromosome.Alternatively, the hlyB and hlyD genes can be cloned on a mobile vector,e. g. plasmid, also encoding the heterologous proteins of interest as afusion with the HlyA signal sequence. As another option, the hlyB andhlyD genes and the fusion between the gene encoding the heterologousprotein of interest and hlyA, can be cloned on separate, compatibleplasmids.

The secreted proteins are useful for the production of protein arrays bytheir binding onto a carrier, optionally via an affinity tag. As anadditional or alternative option, an epitope tag sequence may beincluded for easy detection of the secreted protein of interest.Moreover, in order to avoid loss of function of the protein of interest,optionally it might be useful to remove the HlyA-secretion signal andtag sequences by protease treatment. Therefore, as an option, thecorresponding affinity and/or epitope tag sequences and proteaserestriction sites shall be cloned downstream of the heterologous gene ofinterest.

In connection with the invention it is an option to introduce thehemolysin secretion apparatus into a Gram negative bacterial straincarrying a secA mutation. This is of particular advantage in connectionwith the secretion of proteins which are usually secreted by theSec-dependent secretion pathway and have an N-terminal signal sequencewhich are only inefficiently transported by the hemolysin secretionsystem [Gentschev et al. (1997) Behring Inst. Mitt. 98:103–113].

As an option, the secreted proteins may be used for the production ofprotein arrays with dots of single proteins, preferentially with dots ofequal amounts of single proteins, the latter of which may be acchlevedby binding of the proteins of interest onto a carrier, optionally via anaffinity tag which is able to bind to a suitable partner spotted inequal concentrations on each spot of the array. In case of the doublefilter technology, saturated binding of the affinity tag bound to aprotein of interest secreted from a host cell growing on the upperfilter to its binding partner on the lower filter will ensure binding ofequal amounts of heterologous proteins to the lower filter.Altematively, in case of using a system consisting of differentfilter-separated compartments containing liquid bacterial cultures inone compartment and secreted protein solutions in the other compartment,as it can be realized e. g. in form of a stacked microwell technology,the concentrations of the secreted proteins collected in the bottomwells may be quantified. As an additional or alternative option, anepitope tag sequence may be included for easy detection of the secretedprotein of interest. Moreover, in order to avoid loss of function of theprotein of interest, optionally it might be useful to remove theHlyA-secretion signal and tag sequences by protease treatment.Therefore, as an option, the corresponding affinity and/or epitope tagsequences and protease restriction sites shall be cloned on the vectorencoding the heterologous gene of interest.

a) Construction of a Gram Negative Bacterial Strain With a ChromosomallyEncoded Hemolysin Secretion Apparatus

Another subject of the invention is the construction of a Gram-negativebacterial strain, optionally an E. coli strain, harbouring the hemolysinsecretion system genes hlyB and hlyD within the bacterial chromosome.

In order to create an E. coli strain which stably expresses theα-hemolysin secretion apparatus the hlyB and hlyD genes can beintegrated into the bacterial chromosome of an E. coli strain, inparticular into the secA E.coli mutant strain KL320. Therefore, asexplained in greater detail in the experimental section, plasmidpLacHlyBD may be constructed by the following cloning procedure: The□-pir-dependent suicide plasmid pJRLacZins, (gift of Joachim Reidl,University of Würzburg), containing an EcoRV restriction site within acloned lacZ gene, is digested with the restriction enzyme EcoRV.Consecutively, the hlyBD genes are amplified by PCR from cosmid pCOS10and ligated with the EcoRV restricted plasmid pJRLacZins to createplasmid pLacHlyBD. The plasmid encoded lacZ gene is destroyed duringthis cloning procedure. Plasmid pLacHlyBD allows the integration of thehlyBD into the chromosomal lacZ gene of any E. coli strain or otherbacterial strain harbouring a homologous lacZ gene. The chromosomal lacZgene in E. coli strains not possessing the p-protein, encoded by the pirgene, is being destroyed following the introduction of plasmid pLacHlyBDand a double-crossover event between the lacZ sequences on plasmidpLacHlyBD and the chromosomal lacZ gene, in consequence of which, thehlyB and hlyD genes are being inserted into the chromosomal lacZ gene.

b) Construction of a Plasmid Carrying a Gene Fusion Between a GeneEncoding a Protein of Interest and the 3′-end of HlyA, Encoding theSignal Sequence of the HlyA Protein

A still further subject of the invention is a plasmid carrying a genefusion between a gene encoding a protein of interest and the 3′-end ofhlyA, encoding the signal sequence of the HlyA protein, which allows theexport of the fusion protein, and optionally carrying a proteasecleavage site, affinity tag and/or epitope tag.

As mentioned above, proteins of interest can be made competent forsecretion via the α-hemolysin secretion system by the creation of genefusions between the respective heterologous genes, coding for theproteins of interest, and the C-terminal signal sequence of hlyA. Thegene fusions can be cloned on suitable mobile vectors, includingplasmids, and can consecutively be introduced into a Gram negativebacterial strain which stably expresses the complete α-hemolysinsecretion apparatus.

Spreng and Gentschev (1998) [Spreng and Gentschev (1998) FEMS Microbiol.Left. 165:187–192; Spreng et al. (1999) Mol. Microbiol. 31:1589–1601]showed that the efficacy of heterologous protein secretion via thehemolysin secretion system correlates with the size of the heterologousgene in front of the HlyA_(s) signal. As in the present invention, theHlyA_(s)-fusion protein secreting bacterial strains shall optionally beused for the creation of protein arrays, harbouring equal amounts ofproteins dots, as an option, along with the cloning of the heterologousgene of interest, the cloning of an affinity tag (e. g. C-terminaldomain of the hnRNA-binding protein A1; [Cartegni et al. (1996) J. Mol.Biol. 259:337–348]) in front of the HlyA_(s) signal, which is able tobind to a suitable partner (e. g. single stranded nucleic acids in caseof A1) spotted in equal concentrations on each spot of the array, may beincluded. In case of the double filter technology, saturated binding ofthe affinity tag to its partner on the lower filter will ensureconcomitant binding of equal amounts of heterologous proteins.Alternatively, in case of using a system consisting of differentfilter-separated compartments containing liquid bacterial cultures inone compartment and secreted protein solutions in the other compartment,as it can be realized e. g. In form of a stacked microwell technology,the concentrations of the secreted proteins collected in the bottomwells may be quantified. Moreover, as another option, the cloning of anepitope-tag (e. g. His-tag) can be included, facilitating the detectionof the resulting fusion protein. As another option, suitable proteasecleavage sites should be introduced which allow the removal of thesecretion signal and tag sequences that might affect the function of theprotein of interest.

c) Construction of a Gram Negative Bacterial Strain Which is Able toSecrete a Heterologous Protein of Interest

A still further subject of the invention is the construction of aGram-negative bacterial strain, optionally an E. coli strain, harbouringthe hemolysin secretion system genes hlyB and hlyD within thechromosomal lacZ gene transformed with a plasmid carrying a gene fusionbetween a gene encoding a protein of interest and the 3′-end of hlyA,encoding the HlyA protein which allows the export of the fusion proteinand optionally carrying a protease cleavage site, affinity tag and/orepitope tag.

Following the construction of the vector harbouring a fusion between theprotein of interest and the C-terminal Hly_(s) sequence as well asoptionally also an affinity tag and/or an epitope tag and a proteasecleavage site, transformation of the above describedα-hemolysin-secretion apparatus harbouring host strain results in thefinal bacterial construct.

Eukaryotic System: Construction of Pichia pastoris Strains forHeterologous Protein Secretion

The above described P. pastoris secretion system can be used inconnection with the present invention. In analogy to the above describedE. coli hemolysin secretion system, plasmids derived from the abovementioned Pichia pastoris vectors can be constructed, harbouring variousgenes of interest and optional various protease cleavage sites and tagsequences.

For the production of proteome arrays which are supposed to harbour eachprotein of a certain organism, each protein of this organism shall besecreted. Therefore, for each protein to be secreted, each of thecorresponding genes has to be fused either with the C-terminal signalsequence of hlyA or the α-factor signal and cloned onto a separatevector (e. g. plasmid) which has to be transformed into either theα-hemolysin secretion apparatus expressing bacterial strain or into aPichia pastoris strain. I. e. the total amount of open reading frames ofa certain organism, the proteome of which shall be investigated, willreflect the amount of bacterial constructs required for full coverage ofa proteome of a certain organism.

Utility

Consequenty, this technology allows in particular the production ofarrays harbouring a partial or a complete proteome of an organism forexample as an array of single spots, each of which can be saturated witha single protein for example in an undenatured form or as an alternativethe production of solutions consisting of specific secreted, filteredproteins. Such proteins in particular such arrays can be—directlywithout further purification of the proteins—used for the identificationof protein binding partners of different chemical nature, includingother proteins, nucleic acids, lipids, carbohydrates or other ligands.In case of the production of array-bound proteins, studies with solublebinding partners can be performed directly on the respective array (e.g. filter). In case of the production of protein solutions, bindingstudies can be performed either with array-bound binding partners orwith soluble binding partners. Thus, such array is applicable for allconceivable binding studies, including antibody binding studies andstudies on the binding of pharmaceutical compounds with array-boundtarget proteins. In addition, the impact of pharmaceutical compounds onthe binding behaviour of other molecules can be studied. Moreover, incase of the production of protein solutions, the respective proteins canbe utilized for numerous studies. E. g. they can be analyzed for theirstructural nature and biochemical function, e. g. in enzymatic testsystems, or can be used as antigens for the creation of specificantibodies.

As the array is suitable for testing pharmaceutical drug candidates fortheir protein binding capacities and their impact on the binding andenzymatic capacities of other molecules, the present invention isvaluable for the elucidation of mechanisms of action of the respectivepharmaceutical compounds.

For the production of proteome arrays which are supposed to harbour eachprotein of a certain organism, each protein of this organism shall besecreted. Therefore, for each protein to be secreted, each of thecorresponding genes has to be fused either with an appropriate signalsequence as described above (e. g. E. coli HlyA_(s)- or the yeastα-factor) and cloned onto a separate mobile vector (e. g. plasmid) whichhas to be transformed into the appropriate secretion system carryinghost cell. i. e. the total amount of open reading frames of a certainorganism, the proteome of which shall be investigated, will reflect theamount of bacterial constructs required for full coverage of a proteomeof a certain organism. It is conceivable to create two kinds of thosevectors for each proteome to be investigated: (i) vectors encoding anaffinity tag for retainment of the fusion proteins on a protein arrayand (ii) vectors encoding an epitope tag for facilitated detection ofthe fusion proteins. Thus, studies on the interaction of proteinsproduced by a certain organism could be performed by binding allproteins, encoded on an “affinity tag-vector” via affinity tag onto anarray, overlaying this array with a solution containing a proteinencoded on an “epitope tag-vector” and detecting the interaction betweenthe array-bound and soluble proteins via the epitope tag.

EXPERIMENTAL

Material—M dia, Chemicals, Enzymes, and Ligonucleotides

Bacteria were grown either in liquid Luria-Bertani (LB) or Minimal M9medium, the latter of which was supplemented with 1% casaminoacids or onLB-agar or M9+1% casaminoacids-agar plates or either on Columbla-agarplates with sheep-blood or on enterohemolysin-agar plates with blood,the two latter of which were provided by Oxold (Wesel, Germany). Strainscarrying recombinant plasmids were cultivated under selective antibioticpressure. The antibiotic concentrations were dependent on the copynumber of the respective plasmids (10 to 100 μg/ml). In someexperiments, sucrose at a concentration of 5% and/or 10 ml/l 2% X-Gal(5-Bromo4Cholor-3-indolyl-β-galaktoside in dimethyl formamide) solutionwas added to the bacterial growth medium. Chemicals were purchased byMerck (Ismaning, Germany), Quiagen (Hilden, Germany), Promega (Mannheim,Germany) or Sigma (Deisenhofen, Germany) unless stated otherwise.Restriction enzymes were purchased by Amersham-Pharmacia (Freiburg,Germany). Taq DNA polymerase and other chemicals used for polymerasechain reactions (PCR) were obtained from Gibco BRL, (Karlsruhe,Germany), Boehringer (Ingelheim, Germany) or Eurogentec (Köln, Germany).The oligonucleotides used as primers for PCR are purchased frominteractiva (Ulm, Germany).

Bacterial Strains, Pichia pastoris Strains, Plasmids, Cosmids,Oligonucleotides and Antibodies

The Pichia pastods strains are commercially available from invitrogen.The oligonucleotides used as primers for PCR are listed in Table 1 andwere purchased from interactiva (Ulm, Germany). Antibodies were obtainedfrom different sources and are stated in the results section.

Filter Material and Filter Apparatus

The membrane filters used in the preliminary experiments of thisinvention were provided by Millipore (Eschborn, Germany) or Sartorius(Göttingen, Germany). They consisted either of nitocellulose, mixedcellulose esters or polyvinylidendifluorid. The filters used for acertain experiment are mentioned in the results section.

The “MultiScreen Assay System” provided by Millipore (Eschborn, Germany)was used for the proof of principle of the “stacked microwellstechnology”.

Meth Ds

Determination of Hemolysin Production of Various E. coli Strains

The hemolytic activity of E. coli strains was tested by cultivation ofthose strains on blood agar plates provided by Oxoid. Hemolytic E. colistrains elicited a clear hemolytic zone around the bacterial colonies(Mühidorfer et al., 1996).

The uropathogenic E. coli strain 536, its isogenic mutant 536–21 whichhas lost the pathogenicity islands (Pais) I and II, each of whichcarries a hemolysin operon, [Berger et al. (1982) J. Bacteriol.152:1241–1247; Blum et al. (1994) Infect. Immun. 62:606–614; Hacker etal. (1983) J. Bacteriol. 154:11145–1154], the hly-negative mutant536–39–192 [deposited with Jörg Hacker at the University of Würzburg,Germany and with Inge Mühidorfer, Department of Molecular Biology at BykGulden, Konstanz, Germany] and 15 other E. coli strains (K-12 wt[deposited with the American Typ Culture collection (ATCC) no. 29425],JM109 [deposited with ATCC no. 53323], DH1 [deposited with ATCC no.33849], LE392 [deposited with ATCC no. 33572], W678 [deposited with theGerman strain collection (DSM) no. 6968], 35 [Smith and Linggood (1971)J. Med. Microbiol. 4:467–485], J53 [Taylor (1983) Microbiol. Rev.47:46–83.], EN99 [Blum. (1994) Dissertation, University of Würzburg,Germany] WK6 [deposited with ATCC no. 47078], HB101 [deposited with ATCCno. 33694], 5K [deposited with the E. coli Genetic Stock Center (CGSC)no. 5581], CC118 [Manoil and Beckwith (1985) Proc. Nati. Acad. Sci USA82:8129–8133], C600 [deposited with ATCC no. 23724], MC1029 [Casadabanand Cohen (1980) J. Mol. Biol. 138:179–207], DH5α [deposited with CGSCno. 7855] all of which were provided by Jörg. Hacker, University ofWürzburg, Germany) were compared for their hemolytic activities on sheepblood agar plates following overnight growth. As shown in FIG. 3, onlythe wild-type UPEC strain 536 elicits hemolysis of erythrocytes. In afurther experiment, the E. coli strains 536, 536–21, J53(pUCHlyCA) andJ53(pUC18) were compared for their hemolytic activities on sheep bloodagar plates following overnight growth. Only E. coli 536 andJ53(pUCHlyCA) elicited hemolysis of erythrocytes.

DNA Cloning Techniques

DNA isolation, DNA cleavage with restriction enzymes, agarose gelelectrophoresis, elution of DNA fragments from agarose gels,transformation of E. coli strains with plasmid DNA, generation of DNAprobes, colony and Southern hybridization, and PCR are performed asdescribed in “Molecular Cloning, A Laboratory Manual” [Sambrook andRussell, eds. (2001) 3^(rd) ed. Cold Spring Harbor Laboratory Press, NewYorkl] and/or according to the manuals provided by the above mentionedsuppliers of the corresponding kits, chemicals and enzymes.

DNA Sequencing

Sequencing of DNA, which had been isolated according to the Quiagenprotocol, was performed by GATC (Konstanz, Germany), or TopLab (Munich,Germany).

Cosmid pCOS10 [Knapp et al. (1986) J. Bacteriol. 168:22–30], kindlyprovided by G. Blum-Oehler, University of Würzburg, contains Pai I ofthe uropathogenic E. coli (UPEC) strain 536 [Berger et al. (1982) J.Bacteriol. 152:1241–1247; Blum et al (1994) Infect. Immun. 62:606–614;Hacker et al. (1983) J. Bacteriol. 154:11145–1154]. Following itsisolation from E. coli strain HB101(pCOS10), seqencing of the hemolysinoperon encoded on pCOS10 was performed by GATC (Konstanz, Germany).

PAGE and Western Blot Analysis

Protein detection is acchieved by sodium dodecyl sulfate-polyacrylamidegel electrophoresis (PAGE) and consecutive Western blot analysis, usingspecific primary antibodies and the Promega Proto Blot alkalinephosphatase system [Sambrook and Russell, ads. (2001) Molecular Cloning,A Laboratory Manual, 3^(rd) ed. Cold Spring Harbor Laboratory Press, NewYorkl; Promega manual].

Construction of an E. coli Strain With a Chromosomally Encoded HemolysinSecretion Apparatus

In order to create an E. coli strain which stably expresses theα-hemolysin secretion apparatus, a 3.6 kb DNA fragment encoding the hlyBand hlyD genes was integrated into the bacterial chromosome of variousapathogenic E. coli strains, including K-12 wild-type, E. coli BL21, andJ53.

The following cloning procedure was persued: Plasmid pJRLacZins, (a kindgift of Joachim Reidi, University of Würzburg), was used as a vectorplasmid. It is a λ-pir-dependent suicide plasmid based on plasmidpCVD442, conferring ampicillin resistance due to the presence of the blagene and sensitiveness against growth in the presence of 5% sucrose at agowth temperature of 30° C. due to the presence of the sacB gene.Consequently, plasmid pCVD442 and its derivatives can only replicate inbacteria harbouring the Pir-protein, such as in the E. coli K-12 strainsSy327 and Sm10λpir. Plasmid pJRLacZins, which contains an EcoRVrestriction site within a cloned lacZ gene, was digested with therestriction enzyme EcoRV. Consecutively, the hlyBD genes were amplifiedby PCR from cosmid pCOS10, using the primers StuHlyB1 and HlyD2Stu, andfollowing restriction of the PCR product with Stul, were ligated withthe EcoRV restricted plasmid pJRLacZins to create plasmid pLacHlyBD. Theplasmid encoded lacZ gene was destroyed during this cloning procedure.

Plasmid pLacHlyBD allows the integration of the hlyBD genes into thechromosomal lacZ gene of any E. coli strain or other bacterial strainharbouring a homologous lacZ gene, as it was exemplified with the E colistrains K-12 wild-type, BL21, and J53. The chromosomal lacZ gene in E.coli strains not possessing the p-protein, encoded by the pir gene, isbeing destroyed following introduction of plasmid pLacHlyBD and adouble-crossover event between the lacZ sequences on plasmid pLacHlyBDand the chromosomal lacZ gene, in consequence of which, the hlyB andhlyD genes is being inserted into the chromosomal lacZ gene.lacZ-negative mutants were phenotypically screened for by testing theresulting strains for growing as white colonies on agar platescontaining Luria Bertani medium supplemented with 5% sucrose and X-Galat 30° C. and for loss of ampicillin resistance. Genotypically, themutants were tested by PCR analysis using the primers lacZEV-up andlacZEV-down. The PCR products received from the mutants differed fromthose of the wild-type lacZ-positive strains by size, resulting from theintegration of the hlyB, hlyD sequences. Moreover, the chromosomal DNAof the putative mutants can be restricted with a restriction enzyme thatdoes not cut within the lacZ and integrated hlyBD sequences, and canthen be subjected to Southern blot analyis using a lacZ probe. Mutantsdiffer from the wild-type lacZ-positive strains by a DNA band shift,resulting from the integration of the hlyBD sequences.

Construction of a Plasmid Harbouring a Fusion Between Genes Encoding aProtein X-HlyA_(s)-fusion Protein

In order to experimentally validate the process for the production ofproteins claimed in this invention, we constructed plasmid pUCHlyCAharbouring the hlyC and hlyA genes, which had been amplified by PCR,using the primers StuHlyC4 and HlyA3Stu and cosmid pCOS10 as a template,and following restriction of the PCR product with Stul, were ligatedwith the Smal digested plasmid vector pUC18.

Moreover, plasmid pTOPOMycHisHlyA_(s) was constructed. The genesencoding thrombin, Myc-epitope and His-tag were amplified by PCR usingthe invitrogen plasmid vector pBAD/Myc-HisA as a template and the primerThromMycBAD which harbours the 18 bp thrombin gene sequence as a forwardprimer, and the HispBAD primer as a reverse primer. Cloning of theresulting 89 bp PCR product into the lacZ gene of the invitrogen vectorpCR2.1.-TOPO, resulted in plasmid pTOPO-Thr-Myc-His. Consecutively,hlyA_(s) specific sequences were amplified from pCOS10 using either theprimer pairs XbaHlyA_(s)-up and XbaHlyA_(s)-down or XbaHlyA_(s)-up andXbaHlyA_(s)linker, resulting in hlyAs-specific PCR products with either183 or 99 hlyA_(s)-specific bp and added Xbal sites, which were clonedinto the Xbal site of the above described plasmid pTOPO-Thr-Myc-His. Thefinal plasmid constructs were termed pTOPOMycHisHlyA_(s-)A or -B,harbouring either the 183 or 99 hlyA_(s)-specific bp, respectively.

Construction of a Gram Negative Bacterial Strain Which is Able toSecrete a Heterologous Protein of Interest

In order to experimentally validate the process for the production ofproteins according to the invention, plasmid pUCHlyCA was introducedinto the above described hlyBD+ E. coli constructs by transformation.The resulting E. coli strains harbour a chromosomally encoded hemolysinsecretion system and a plasmid encoded cytotoxin, the α-hemolysin HlyA,and toxin activator HlyC. Secretion of the HlyA cytotoxin became obviousby the hemolytic activity of the sterile-filtered (using 0,45 μmfilters) culture supernatant on sheep blood agar plates.

Construction of E. coli α-hemolysin Based Secretion Systems

As described above, the hlyB and hlyD genes were introduced into theGram-negative bacterial genome, harbouring a chromosomally encoded tolCgene, in order to enable the bacteria to express the completeα-hemolysin secretion apparatus. Proteins of interest were madecompetent for secretion via the α-hemolysin secretion system by thecreation of gene fusions between the respective heterologous genes ofinterest and the C-terminal signal sequence of HlyA (hlyA_(s)). The genefusions were cloned on suitable mobile vectors, such as plasmids orcosmids. Consecutively, they were introduced into a Gram negativebacterial strain which expresses the complete α-hemolysin secretionapparatus, consisting of HlyB, HlyD and TolC. The stable expression ofthe secretion system could be acchieved by insertion of the hlyB andhlyD genes into the bacterial chromosome. Alternatively, the hlyB andhlyD genes can be cloned on a mobile vector, e. g. plasmid, alsoencoding the heterologous proteins of interest as a fusion with the HlyAsignal sequence, or the hlyB and hlyD genes and the fusion between thegene encoding the heterologous protein of interest and hlyA_(s) can becloned on separate, compatible plasmids.

In order to create an E. coli strain which stably expresses theα-hemolysin secretion apparatus, a 3.6 kb DNA fragment encoding the hlyBand hlyD genes was integrated into either of the chromosomal genes lacZor recA gene of E. coli by the following cloning procedures:

Integration of hlyB and hlyD Into the Chromosomal lacZ Gene of E. coliStrain J53

The 9.5 kb plasmid pJRLacZins, (a kind gift of Joachim Reidl, Universityof Würzburg), was used as a vector plasmid. It is a λ-pir-dependentsuicide plasmid based on the 6.3 kb plasmid pCVD442, conferringampicillin resistance due to the presence of the bla gene andsensitiveness against growth in the presence of 5% sucrose at a gowthtemperature of 30° C. due to the presence of the sacB gene.Consequently, plasmid pCVD442, the DNA sequence of which was provided bythe company GATC, Konstanz, Germany, and its derivatives can onlyreplicate in bacteria harbouring the λ-protein, such as in the E. coliK-12 strains Sy327 and Sm10λpir. Plasmid pJRLacZins, which contains anEcoRV restriction site within the lacZ sequence, was digested with therestriction enzyme EcoRV. Consecutively, a 3.6 kb DNA fragmentcontaining the hlyB and hlyD genes was amplified by PCR from cosmidpCOS10, using the forward primer5′-AAAAGCCCTTTTATGGATTCTTGTCATAAAATTGATTATGGG (SEQ ID NO: 24),designated Stu-HlyB1 (designed according to hemolysin-specific sequencewith accession no. M14107), and the reverse primer5′-AAAAGGCCTTTTTTAACGCTCACGTAAACTTTCTGT (SEQ ID NO: 25), designatedHlyD2Stu (designed according to hemolysin-specific sequence withaccession no. M14107), and following restriction of the PCR product withStuI, ligated with the EcoRV restricted plasmid pJRLacZins to create the13.1 kb plasmid pLacHlyBD. The plasmid encoded lacZ gene was destroyedduring this cloning procedure. Plasmid pLacHlyBD allows the integrationof the hlyBD genes into the chromosomal lacZ gene of any E. coli strainor other bacterial strain harbouring a homologous lacZ gene. Thechromosomal lacZ gene in E. coli strains not possessing the ρ-protein,encoded by the pir gene, was destroyed following introduction of plasmidpLacHlyBD and a double-crossover event between the lacZ sequences onplasmid pLacHlyBD and the chromosomal lacZ gene, in consequence ofwhich, the hlyB and hlyD genes were inserted into the chromosomal lacZgene. LacZ-negative mutants were phenotypically screened by testing theresulting strains for growing as white colonies on agar platescontaining Luria Bertani medium supplemented with 5% sucrose and X-Galat 30° C. and for loss of ampicillin resistance. Genotypically, themutants were tested by PCR analysis using the E. coli lacZ specific(accession no. V00296) forward primer lacZEV-up(5′-CTGCTGCTGCTGAACGGCAAG) (SEQ ID NO: 31), and reverse primerlacZEV-down (5′-TCATTGGCACCATGCCGTGGG) (SEQ ID NO: 32). The PCR productsreceived from the mutants differed from those of the wild-typelacZ-positive strains by size, resulting from the integration of thehlyB and hlyD sequences. Moreover, insertion of the hlyB and hlyDsequences into the chromosomal lacZ gene was assured by DNA sequencing.

Integration of hlyB and hlyD Into the Chromosomal RecA Gene of E. coliK-12 Strain J53

A 3.6 kb DNA fragment containing the hlyB and hlyD genes was amplifiedby PCR using the cosmid pCOS10 as a template and the forward primerMibi-151 (5′-ATGGATTCTTGTCATAAAATTGATTATGGG) (SEQ ID NO: 1) and thereverse primer HlyD2 (5′-TTAACGCTCACGTAAACTTTCTGT) (SEQ ID NO: 21),designed according to hemolysin-specific sequence of cosmid pCOS10 whichwas established by the company GATC, Konstanz, Germany), and accessionno. M14107, respectively. The resulting 3.6 kb PCR product was ligatedwith the 3.5 kb EcoRV restricted plasmid vector pETBlue-1 which iscommercially available from the company Novagen Calbiochem-Novabiochem,Bad Soden, Germany, resulting in the 7.1 kb plasmid pETBlue1-hlyBD. Inconsequence, expression of the hlyB and hlyD genes was under control ofthe IPTG-inducible T7 promoter.

PCR using plasmid pETBlue1-hlyBD as a template and the forward primerMibi-169 (5′-CTAACCTGACCTAAAATTGTGAGCGCTCACAATTCTCGTGA) (SEQ ID NO: 2),designed according to pETBlue-1-hlyBD sequence, and the above describedreverse primer HlyD2, resulted in a 3.9 kb PCR product, designated“Stop-lacO/T7 promoter-hlyBD” harbouring the hlyB and hlyD genes as wellas stop codons and lacO/T7 promoter sequences upstream of hlyB.

PCR using the E. coli K-12 strain C600 as a template and theygaD-specific forward primer Mibi-167 (5′-ATGACTGACAGTGAACTGATGCAG) (SEQID NO: 3) and the oraA-specific reverse primer Mibi-168(5′-TCAGTCGGCAAAATTTCGCCAAATCTCC) (SEQ ID NO: 4), both designedaccording to accession no. AE000354, resulted in the amplification of a2.2 kb DNA fragment harbouring the recA gene flanked upstream by ygaDand downstream by oraA sequences. Insertion of the resulting 2.2 kb PCRproduct into position 295 of the 3.9 kb plasmid vector pCR2.1-TOPO,which is commercially available from the company Invitrogen Corporation,resulted in the 6.1 kb plasmid pCR2.1-TOPO-ygaD-recA-oraA. PlasmidpCR2.1-TOPO-ygaD-recA-oraA was linerarized by digestion with therestriction enzyme AccI, which cuts at position 994 of the 2.2 kbygaD-recA-oraA insert within the recA gene, and blunt-ended by treatmentwith Klenow fragment.

Ligation of the above described 3.9 kb PCR product “Stop-lacO/T7promoter-hlyBD” with the above described Accl restricted 6.1 kb plasmid“pCR2.1-TOPO-ygaD-recA-oraA” resulted in the 10 kb plasmid“pCR2.1-ygaD-recA′-Stop-P_(T7)-hlyBD-recA″-oraA”, which wasconsecutively digested with the restriction enzymes Sacl and Sphl,resulting in a 6.2 kb DNA fragment containing the 3.9 kb “Stop-lacO/T7promoter-hlyBD” fragment flanked upstream by ygaD and recA specificsequences and downstream by recA and oraA specific sequences.Consecutively, this 6.2 kb DNA fragment was ligated with the Sacl andSphl restricted above described 6.3 kb suicide vector plasmid pCVD442,resulting in the 12.5 kb plasmid pCVD442-P_(T7)-hlyBD.

Plasmid pCVD442-P_(T7)-hlyBD allowed the integration of the hlyBD genesunter control of the T7 promoter into the chromosomal recA gene of recA₊E. coli strains. The chromosomal recA gene in E. coli strains notpossessing the ρ-protein, encoded by the pir gene, was destroyedfollowing introduction of plasmid pCVD442P_(T7)-hlyBD and adouble-crossover event between the recA sequences on plasmidpCVD442-P_(T7)-hlyBD and the chromosomal recA gene, in consequence ofwhich, the hlyB and hlyD genes were inserted into the chromosomal recAgene. RecA-negative mutants were phenotypically screened for by testingthe resulting strains for their growth on agar plates containing LuriaBertani medium supplemented with 5% sucrose and X-Gal at 30° C., forloss of ampicillin resistance, as well as for sensitivity to UV light,mitomycin C and methylmethanethiosulsfonate [Mühldorfer et al. (1996)Infect. Immun. 64:495–502]. Genotypically, the mutants were tested byPCR analysis using E. coli recA specific (accession no. AE000354)forward primer Mibi-224 (5′-CGCTGACGCTGCAGGTGATCGCCG) (SEQ ID NO: 5) andreverse primer Mibi-225 (5′-TCCGGGTTACCGAACATCACACCA) (SEQ ID NO: 6)binding up- and downstream of the AccI site within the recA gene,respectively. The PCR products received from the mutants differ fromthose of the wild-type recA-positive strains by size, resulting from theintegration of the hlyB and hlyD sequences. Moreover, insertion of thehlyB and hlyD sequences into the chromosomal recA gene was assured byDNA sequencing.

Construction of Plasmids Harbouring Fusions Between Genes Encoding aProtein of Interest and HlyA_(s).

As mentioned above, proteins of interest were made competent forsecretion via the α-hemolysin secretion system by creating gene fusionsbetween the respective heterologous genes of interest and the C-terminalsignal sequence of HlyA (hlyA_(s)). The gene fusions were cloned onsuitable mobile vectors, such as plasmids or cosmids. Consecutively,they were introduced into a Gram negative bacterial strain whichexpresses the complete α-hemolysin secretion apparatus, consisting ofHlyB, HlyD and TolC. In addition to the fusion between the gene ofinterest and hlyA_(s), as an option, sequences encoding affinity tagand/or epitope tag and/or protease cleavage site may be included asdescribed above. The stable expression of the secretion system could beacchieved by insertion of the hlyB and hlyD genes into the bacterialchromosome. Alternatively, the hlyB and hlyD genes can be cloned on amobile vector, e. g. plasmid, also encoding the heterologous proteins ofinterest as a fusion with the HlyA signal sequence. As another option,the hlyB and hlyD genes and the fusion between the gene encoding theheterologous protein of interest and hlyA_(s) can be cloned on separate,compatible plasmids.

Plasmids harbouring fusions between hlyA_(s) and various heterologousgenes of interest, which are subsequently transformed into E. colistrains harbouring either chromosomal or plasmid located hlyBD as wellas a chromosomal tolC gene, were constructed. Examples herefore are theplasmid constructs pUCHlyCA, pETBlue-1-HlyCA, pETBlue-1-PhoA-HlyAs andpETBlue-1-lacZ-HlyAs, which were created as follows:

A 3.6 kb DNA fragment was amplified by PCR using the forward primerStuHlyC4 (5′-AAAAGGCCTTTTATGAATATAAACAAACCATTAGAG) (SEQ ID NO: 22) andreverse primer HlyA3Stu (5′-AAAAGGCCTTTTTTATGCTGATGTGGTCAGGGTTATTGAG)(SEQ ID NO: 23), designed according to hemolysinspecific sequences withaccession no. M14107 and StuI digestable sequences, and cosmid pCOS10 asa template. The resulting PCR product was digested with the restrictionenzyme StuI and consecutively ligated with the SmaI digested plasmidvector pUC18 to create the 6.3 kb plasmid pUCHlyCA, harbouring the hlyCand hlyA genes.

A 3.6 kb DNA fragment was amplified by PCR using the forward primerMibi-142 (5′-ATGAACAGAAACAATCCATTAGAGGTTCTT) (SEQ ID NO: 7) and reverseprimer Mibi-143 (5′-TTATGCTGATGCGGTCAAAGTTATTGAGTTCCG) (SEQ ID NO: 8),designed according to hemolysin-specific sequence of cosmid pCOS10 whichwas established by the company GATC, Konstanz, Germany). The resultingPCR product was ligated with the EcoRV digested 3.5 kb plasmid vectorpETBlue-1 to create the 7.1 kb plasmid pETBlue-1-HlyCA, harbouring thehlyC and hlyA genes under control of the T7 promoter.

A 1.5 kb DNA fragment was amplified in a 3-step crossover PCR using E.coil as a template and the phoA specific forward primer Mibi-148(5′-ATGCGGACACCAGAAATGCCTGTTCTGGAA) (SEQ ID NO: 9) and the phoA specificreverse primer Mibi-226 (5′-TTTCAGCCCCAGAGGGGCTTTCAT) (SEQ ID NO: 10) inthe fist PCR, pCOS10 as a template and the phoA/hlyAs specific forwardprimer Mibi-149 (5′-AAAGCCGCTCTGGGGCTGAAATCAACTTATGCAGACCTGGAT ) (SEQ IDNO: 11) and the reverse primer Mibi-145(5′-TTATGCTGATGCGGTCAAAGTTATTGAGTT) (SEQ ID NO: 12) in the second PCRand the resulting PCR products from the first and second PCRs astemplates and the primers Mibi-148 and Mibi-145 in the 3^(rd) PCR. Therespective primers were designed according to E. coli phoA (accessionno. M29666 J04079) and hemolysin specific sequences of cosmid pCOS10which was established by the company GATC, Konstanz, Germany). Theresulting 1.5 kb PCR product from the third PCR was ligated with theEcoRV digested 3.5 kb plasmid vector pETBlue-1 to create the 5 kbplasmid pETBlue-1-PhoA-HlyAs, harbouring the phoA-hiyA_(s) fusion undercontrol of the T7 promoter.

A 3.2 kb DNA fragment was amplified in a 3-step crossover PCR using E.coil as a template and the lacZ specific forward primer Mibi-144(5′-ATGACTATGATTACAGATTCACTGGCCGTC) (SEQ ID NO: 13) and the lacZspecific reverse primer Mibi-227 (TTTTTGACACCAGACCAACTGGTA) (SEQ ID NO:14) in the fist PCR, pCOS10 as a template and the lacZ/hlyAs specificforward primer Mibi-146 (5′-CAGTTGGTCTGGTGTCAAAAATCAACTTATGCAGACCTGGAT)(SEQ ID NO: 15) and the reverse primer Mibi-145(5′-TTATGCTGATGCGGTCAAAGTTATTGAGTT) (SEQ ID NO: 12) in the second PCRand the resulting PCR products from the first and second PCRs astemplates and the primers Mibi-144 and Mibi-145 in the 3^(rd) PCR. Therespective primers were designed according to E. coli lacZ (accessionno. V00296) and hemolysin specific sequences of cosmid pCOS10 which wasestablished by the company GATC, Konstanz, Germany). The resulting 3.2kb PCR product from the third PCR was ligated with the EcoRV digested3.5 kb plasmid vector pETBlue-1 to create the 6.7 kb plasmidpETBlue-1-LacZ-HlyAs, harbouring the lacZ-hlyA_(s) fusion under controlof the T7 promoter.

The 4.2 kb plasmid pTOPO-Thr-Myc-His-HlyA_(s) was constructed asfollows: The genes encoding thrombin, Myc-epitope and His-tag wereamplified by PCR using the Invitrogen plasmid vector pBAD/Myc-HisA as atemplate and the primer ThromMycBAD(5′-CTGGTTCCGCGTGGATCTGGGCCCGAACAAAAACTCATCTCA) (SEQ ID NO: 29) whichharbours the 18 bp thrombin gene sequence as a forward primer, and theprimer HispBAD (5′-TCAATGATGATGATGATGATGGTCGACGGC) (SEQ ID NO: 30) as areverse primer. Cloning of the resulting 89 bp PCR product into the lacZgene of the Invitrogen vector pCR2.1.-TOPO, results in the 4.0 kbplasmid pTOPO-Thr-Myc-His. Consecutively, a 213 bp DNA fragmentcontaining hlyAs flanked by NsiI restrictable sequences were amplifiedusing the primers Mibi-108(5′-CCAATGCATTGGTTCTGCAGTTGTCAACTTATGCAGACCTGG) (SEQ ID NO: 16) andMibi-109 (5′-CCAATGCATTGGTTCTGCAGTTGTTATGCTGATGCGGTCAAA) (SEQ ID NO: 17)and the cosmid pCOS10 as a template. Ligation of the resulting, NsiIrestricted PCR product with the NsiI restricted vector pTOPO-Thr-Myc-Hisresulted in the final 4.2 kb plasmid construct pTOPO-Thr-Myc-His-HlyAs,containing sequences encoding thrombin, Myc-epitope, His-tag upstream ofhlyAs and upstream of single KpnI and BamHI cloning sites which wereused as integration sites for various genes of interest, e. g. hlyCA,lacZ, phoA.

Moreover, plasmids harbouring fusions between hlyA, and variousheterologous genes of interest as well as the hlyB and hlyD genes, whichwere subsequently transformed into E. coli strains only harbouring thechromosomal tolC, were constructed. Examples herefore are the plasmidconstructs pUCHlyCABD, pETBlue-1-PhoA-HlyAsBD andpETBlue-1-lacZ-HlyAsBD. As a control vector, pETBlue-1-HlyAsBD was alsoconstructed.

Plasmid pUCHlyCABD was constructed as follows: A 7.2 kb DNA fragment wasamplified by PCR using the forward primer Mibi-142(5′-ATGAACAGAAACAATCCATTAGAGGTTCTT) (SEQ ID NO: 7) and reverse primerHlyD2 (5′-TTAACGCTCACGTAAACTTTCTGT) (SEQ ID NO: 21), designed accordingto hemolysin-specific sequence of cosmid pCOS10 which was established bythe company GATC, Konstanz, Germany). The resulting PCR product wasligated with the EcoRV digested 3.5 kb plasmid vector pETBlue-1 tocreate the 10.7 kb plasmid pETBlue-1-HlyCABD, harbouring the hlyCABDoperon under control of the T7 promoter.

Plasmid pETBlue-1-PhoA-HlyAsBD was constructed as follows: A 5.2 kb DNAfragment was amplified in a 3-step crossover PCR using E. coli as atemplate and the phoA specific forward primer Mibi-148(5′-ATGCGGACACCAGAAATGCCTGTTCTGGAA) (SEQ ID NO: 9) and the phoA specificreverse primer Mibi-226 (5′-TTTCAGCCCCAGAGCGGCTTTCAT) (SEQ ID NO: 10) inthe first PCR, pCOS10 as a template and the phoA/hlyAs specific forwardprimer Mibi-149 (5′-AAAGCCGCTCTGGGGCTGAAATCAACTTATGCAGACCTGGAT) (SEQ IDNO: 11) and the reverse primer HlyD2 (5′-TTAACGCTCACGTAAACTTTCTGT) (SEQID NO: 21) in the second PCR and the resulting PCR products from thefirst and second PCRs as templates and the primers Mibi-148 and HlyD2 inthe 3^(rd) PCR. The respective primers were designed according to E.coli phoA (accession no. M29666 J04079) and hemolysin specific sequencesof cosmid pCOS10 which was established by the company GATC, Konstanz,Germany). The resulting 5.2 kb PCR product from the third PCR wasligated with the EcoRV digested 3.5 kb plasmid vector pETBlue-1 tocreate the 8.7 kb plasmid pETBlue-1-PhoA-HlyAsBD, harbouring thephoA-hiyA_(s)BD fusion under control of the T7 promoter.

Plasmid pETBlue-1-LacZ-HlyAsBD was constructed as follows: A 6.9 kb DNAfragment was amplified in a 3-step crossover PCR using E. coli as atemplate and the lacZ specific forward primer Mibi-144(5′-ATGACTATGATTACAGATTCACTGGCCGTC) (SEQ ID NO: 13) and the lacZspecific reverse primer Mibi-227 (5′-TTTTTGACACCAGACCAACTGGTA) (SEQ IDNO: 14) in the fist PCR, pCOS10 as a template and the lacZ/hlyAsspecific forward primer Mibi-146(5′-CAGTTGGTCTGGTGTCAAAAATCAACTTATGCAGACCTGGAT) (SEQ ID NO: 15) and thereverse primer HlyD2 (5′-TTAACGCTCACGTAAACTTTCTGT) (SEQ ID NO: 21) inthe second PCR and the resulting PCR products from the first and secondPCRs as templates and the primers Mibi-144 and HlyD2 in the 3^(rd) PCR.The respective primers were designed according to E. coli lacZ(accession no. V00296) and hemolysin specific sequences of cosmid pCOS10which was established by the company GATC, Konstanz, Germany). Theresulting 6.9 kb PCR product from the third PCR was ligated with theEcoRV digested 3.5 kb plasmid vector pETBlue-1 to create the 10.4 kbplasmid pETBlue-1-LacZ-HlyAsBD, harbouring the lacZ-hlyA_(s)BD fusionunder control of the T7 promoter.

Plasmid pETBlue-1-HlyAsBD was constructed as follows: A 3.8 kb DNAfragment was amplified by PCR using the forward primer Mibi-228(5′-TCAACTTATGCAGACCTGGATAAT) (SEQ ID NO: 18) and reverse primer HlyD2(5′-TTAACGCTCACGTAAACTTTCTGT) (SEQ ID NO: 21), designed according tohemolysin-specific sequence of cosmid pCOS10 which was established bythe company GATC, Konstanz, Germany). The resulting PCR product wasligated with the EcoRV digested 3.5 kb plasmid vector pETBlue-1 tocreate the 7.3 kb plasmid pETBlue-1-HlyAsBD, harbouring hlyAsBD undercontrol of the T7 promoter.

Construction of Pichia pastoris α-factor Based Secretion Systems

The above described P. pastoris secretion system is used in a modifiedversion for FunProTec. In analogy to the above described E. colihemolysin secretion system, plasmids derived from the above mentionedinvitrogen Pichia pastoris vectors are constructed, harbouring variousgenes of interest and optional various protease cleavage sites and tagsequences. Plasmids harbouring fusions between the α-factor signalsequence and various heterologous genes of interest are constructedusing the Invitrogen plasmid vectors pPICZalpha.

Plasmid pPICZalpha-A-LacZ is constructed as follows: A 3.1 kb DNAfragment is amplified by PCR using E.coli as a template and the lacZ(accession no. NC 002655) specific forward primer Mibi-159(5′-ATATATGGGGTACCACTATGATTACAGATTCACTGG) (SEQ ID NO: 19), lacking theinitial start codon but harbouring a KpnI cleavable sequence, and thelacZ specific reverse primer Mibi-162(5′-ATATATGCTCTAGATTTTTGACACCAGACCAACTG) (SEQ ID NO: 20), lacking thestop codon but harbouring a XbaI cleavable sequence. The KpnI and XbaIrestricted 3.1 kb PCR product is ligated with the 3.6 kb KpnI and XbaIrestricted Invitrogen plasmid pPICZalphaA, resulting in the 6.7 kbplasmid pPICZalpha-A-LacZ. Following the propagation of this shuttleplasmid in E. coli Top10 F′, the α-factor signal sequence-lacZ fusion isintegrated into the P. pastoris genome after transformation of plasmidpPICZalpha-A-LacZ into P. pastoris and a crossover event between theAOX1 sequences on plasmid pPTCZalpha-A-LacZ and the chromosomal AOX1sequences.

Establishment of a Compartment System for the Preservation of SecretedProteins

Double Filter Technique

As illustrated in FIG. 1, two filters with an appropriate pore size areplaced on top of each other on a bacterial growth agar plate. The topfilter has a pore diameter that prevents bacterial migration from thetop to the lower filter, but allows flow of the secreted fusion proteinsto the lower filter. It has a low protein binding capacity. inconstrast, the lower filter has an optimal protein binding capacity,either by nature or as a consequence of being coated at certain spotswith a component which is an optimal binding partner for a component ofthe protein fusion to be secreted by the bacteria growing on the topfilter. Both filters allow the flow-through of nutrients and growthfactors from the bacterial growth agar plate to the bacterial coloniesgrowing on the top filter. The bacterial colonies expessing a hemolysinsecretion apparatus will secrete HlyA_(s)-protein fusions that willdiffuse onto the lower filter which they bind to.

The above described E. coli strains 536, its isogenic mutants 536–21 and536–39–192, E. coli K-12 wt, JM109, DH1, LE392, W678, 35, J53, EN99,WK6, HB101, 5K, CC118, C600, MC1029, DH5α) are subjected to the doublefilter technology: Two Millipore filters are placed on top of each otheron a bacterial growth agar plate. The top filter (hydrophilic Duraporemembrane filter with a low protein binding capacity, provided byMillipore, Eschbom, Germany, order no. HVLP09050) has a pore diameter of0,45 μm, preventing bacterial migration from the top to the lowerfilter, but allowing flow of the secreted fusion proteins to the lowerfilter. The Millipore MF membrane filter (order no. VCWP09025),consisting of a mixed ester of cellulose acetate and cellulose nitrate,and having an optimal protein binding capacity by its chemical nature,is chosen as the lower filter. Both filters allow the flow-through ofnutrients and growth factors from the bacterial growth agar plate to thebacterial colonies growing on the top filter. The bacteria are pickedonto the top filter and growth is allowed to occur for 3 hours at 37° C.Consecutively, the secreted proteins which bind to the lower filter arevisualized by PonceauS staining. By this method, only protein secretedby E. coli 536 is detectable. In another experiment, the lower filter isplaced onto a sheep blood agar plate. Following overnight incubation atroom temperature, lysis is visable at spots which lay underneath the E.coli 536 secreted protein toxin (FIG. 3). In a further experiment, theE. coli strains 536, 536–21, J53(pUCHlyCA) and J53(pUC18) are subjectedto the double filter system, in consequence of which the lower filter isremoved following 3 hours of bacterial growth at 37° C. and placed ontoa sheep blood agar plate. Following overnight incubation at roomtemperature, lysis is visable at spots which lay underneath the proteinspots derived from E. coli 536 and J53(pUCHlyCA).

Stacked Microwells Technique

As described in FIG. 2, stacked microwell plates are used, consisting ofmicrowells placed on top of each other and separated by a filtermembrane with a pore diameter that prevents bacterial migration butallows flow of the secreted fusion proteins from the top well to thelower well. Liquid cultures of bacteria are growing in the uppermicrowell plate. Proteins secreted by those bacteria diffuse into thelower well, from which they can be harvested as a protein solution. Inparticular it is preferred that, a liquid culture of a bacteriumexpessing a hemolysin secretion apparatus secretes HlyA_(s)-proteinfusions that diffuse into the lower well, from which it can be harvestedas a protein solution. The “MultiScreen Assay System” provided byMillipore (Eschbom, Germany) and the Millipore MultiScreen filtrationplates MAHVS4510 with 0,45 μm Durapore PVDF membranes and the GreinerPolyproyien-Microplates 651201 are used as the lower harvest plates inpreliminary experiments. The above described E. coli strains 536, itsisogenic mutants 536–21 and 536–39–192, E. coli K-12 wt, JM109, DH1,LE392, W678, 35, J53, EN99, WK6, HB101, 5K, CC118, C600, MC1029,subjected to the double filter technology. 20 μl of each of the proteinsolutions harvested from the lower plates are places onto Oxoid sheepblood agar plates. Only the protein secreted by E. coli 536 elicitshemolysis.

Determination of Protein Secretion of E. coli and Pichia pastorisStrains

Protein secretion of E. coli and Pichia pastoris strains can be testedfollowing the application of the double filter technique, describedabove: Following binding to the lower filter in this system the secretedproteins can subsequently be subjected to standard protein detectionmethods, including ink, PonceauS, Coomassie or silver stains.

In case of the HlyA cytotoxin, its secretion can be monitored by itshemolytic activity of the sterile-filtered (using 0,45 μm filters)culture supematant on sheep blood agar plates.

Generally, in case of proteins with a known function, thefilter-sterilized culture supernatants of the respective bacteria can beexamined in functional assays.

EXAMPLES OF RESULTS OBTAINED WITH THE INVENTION

DNA Sequence of the Hemolysin Operon which is Located on thePathogenicity Island I (Pai I) of E coli Strain 536

Cosmid pCOS10 [Knapp et al. (1986) J. Bacterdol. 168:22–30] kindlyprovided by G. Blum-Oehler, University of Würzburg, contains Pai I ofthe uropathogenic E coli (UPEC) strain [Berger et al. (1982) J.Bacteriol. 152:1241–1247; Blum et al. (1994) Infect. Immun. 62:606–614;Hacker et al. (1983) J. Bacteriol. 154:11145–1154]). Following itsIsolation from E. coli strain HB101(pCOS10) by the Quiagen protocoll,this cosmid was used as a template in PCRs with various primer sets toamplify overlapping fragments of the hemolysin operon located on pCOS10.The sequences of the primers were chosen from the DNA sequence of thehemolysin operon encoded by the E. coli plasmid pHlyl 52, which waspublished by Hess et al., 1986, with accession no. M14107.

Hemolytic Activities of Various E. coli Strains

The uropathogenic E. coli strain 536, its isogenic mutant 536–21 whichhas lost the pathogenicity islands (Pais) I and II, each of whichcarries a hemolysin operon [Berger et al. (1982) J. Bacteriol.152:1241–1247; Blum et al. (1994) Infect. Immun. 62:606–614; Hacker etal. (1983) J. Bacteriol. 154:11145–1154], E. coli KL320-HlyBD(pUCHlyCA)and various E. coli K-12 strains were compared for their hemolyticactivities on sheep blood agar plates following overnight growth. Onlythe wild-type UPEC strain 536 and E. coli strain KL320-HlyBD(pUCHlyCA)but not the UPEC mutant 536–21 or any of the other E. coli K-12 strainselicited hemolysis of erythrocytes.

Double Filter Technology on Blood Agar Plate

UPEC strain 536, its mutant 536–21 as well as 15 E. coli K-12 strainsand E. coli KL320-HlyBD (pUCHlyCA) were subjected to the double filtertechnology described above. The upper filter showed all the various E.coli colonies, the PonceauS treated lower filter only displayed staineddots underneath the filter spots of E. coli strain 536 and E. coliKL320-HlyBD(pUCHlyCA). The sheep blood agar underneath the double filtersystem also only showed hemolytic zones at spots which were localizedunderneath the filter carrying E. coli strain 536 and E. coliKL320-HlyBD(pUCHlyCA).

In another experiment, the lower filter was removed following 3 hours ofbacterial growth at 37° C. and placed onto another sheep blood agarplate. Following overnight incubation at room temperature, lysis becameagain visable at spots which lay underneath the E. coli 536 and E. coliKL320HlyBD(pUCHlyCA) secreted protein toxin.

BRIEF DESCRIPTION OF FIGURES

FIG. 1: Double filter technique. Two filters (1, 2) are placed on top ofeach other on a bacterial growth agar plate (3) forming a compartmentsystem (28). Cells secreting proteins of interest (5) are growing on thetop filter (1) in the form of single colonies (4) whereby the growingcells on the top filter (1) form a first compartment (29). A lowerfilter (2) and the bacterial growth agar plate (3) are forming a secondcompartment (30). The top filter (1) which is part of the firstcompartment functions as a barrier which barrier has a pore diameterthat prevents bacterial migration from the top filter (1) to the lowerfilter (2), but allows flow of the secreted proteins of interest (5) tothe lower filter (2), the latter of which is part of the secondcompartment (30) and has an optimal protein binding capacity. Bothfilters (1, 2) allow the flow-through of nutrients and growth factorsfrom the bacterial growth agar plate (3) to the bacterial colonies (4)growing on the top filter (1). In the first compartment (29), growingbacterial cell colonies (4) have a discrete arrangement. Consequently,secreted proteins of interest (5) migrate through the top filter (1)onto the lower filter (2) of the second compartment (30) building anarray of descrete protein spots.

FIG. 2: Stacked mircowells technique. Two microtiter plates (6, 7) areplaced on top of each other and separated by a filter membrane (8)forming a compartment system (28). A liquid culture of cells (9) growingin the wells of the top microliter plate (6) and the mebrane (8) areforming the first compartment (29) of the compartment system (28). Thesecond compartment (30) is constituted by wells of the lower microtiterplate (7) in which proteins of interest (5) are secreted. The filtermembrane (8) has a pore diameter that prevents bacterial migration butallows flow of the proteins of interest (5) from wells of the topmicrotiter plate (6) to the wells of the lower mlcrotiter plate (7). Aliquid culture (9) of cells growing in the wells of the top microtiterplate (6) will secrete proteins of interest (5) which diffuse into wellsof the lower microfiter plate (7), from which they are harvested.

FIG. 3: Double filter system. The E. coli strains 536 (10), 536–21 (11),536–39–192 (12), JM109 (14), DH1 (15), LE392 (16), W678 (17), 35 (18),J53 (19), EN99 (20), WK6 (21), HB101 (22), 5K (23), CC118 (24), C600(25), MC1029 (26) and DH5Δ (27) are subjected to the double filtersystem:

Two Millipore filters (1,2) are placed on top of each other on abacterial growth agar plate (3). The top filter (1) (hydrophilicDurapore membrane filter with a low protein binding capacity, providedby Millipore, Eschbom, Germany, order no. HVLP09050) has a pore diameterof 0,45 μm, preventing bacterial migration from the top to the lowerfilter (2), but allowing flow of the secreted fusion proteins to thelower filter (2). The Millipore MF membrane filter (order no.VCWP09025), consisting of a mixed ester of cellulose acetate andcellulose nitrate, and having an optimal protein binding capacity by itschemical nature, is used as the lower filter (2). Both filters (1,2)allow the flow-through of nutrients and growth factors from thebacterial growth agar plate (3) to the bacterial colonies growing on thetop filter (1). The bacteria are picked onto the top filter and growthis allowed to occur for 3 hours at 370C. Consecutively, the lower filter(2) is removed following 3 hours of bacterial growth at 37° C. andplaced onto a sheep blood agar plate. Following overnight incubation atroom temperature, lysis is visable at spots which lay underneath the E.coli 536 secreted protein toxin.

TABLES TABLE 1. Oligonucleotides used in the present applicationOligonucleotide anneals to gene or designation nucleotide sequenceplasmid position strand HlyD2 5′-TTA ACG CTC ACG TAA ACT TTC TGT-3′hlyD/8044-8021*¹ − (SEQ ID NO:21) StuHlyC4 5′-AAA AGG CCT TTT ATG AATATA AAC AAA hlyC/796-819*¹ + CCA TTA GAG-3′ (SEQ ID NO: 22) HlyA3Stu5′-AAA AGG CCT TTT TTA TGC TGA TGT GGT hlyA/4394-4367*¹ − CAG GGT TATTGA G-3′ (SEQ ID NO: 23) StuHlyB1 5′-AAA AGG CCT TTT ATG GAT TCT TGT CAThlyB/4466-4495*¹ + AAA ATT GAT TAT GGG-3′ (SEQ ID NO: 24) HlyD2Stu5′-AAA AGG CCT TTT TTA ACG CTC ACG TAA hlyD/8044-8021*¹ − ACT TTC TGT-3′(SEQ ID NO: 25) XbaHlyA_(s)-up 5′-CTA GTC TAG ACT ACT TAG CCT ATG GAAhlyA/4212-4239*¹ + GTC AGG GTG ATC TTA-3′ (SEQ ID NO: 26)XbaHlyA_(s)-down 5′-CTA GTC TAG ACT AGT TAT GCT GAT GTG hlyA/4367-4394*¹− GTC AGG GTT ATT GAG-3′ (SEQ ID NO: 27) XbaHlyA_(s)-linker 5′-CTA GTCTAG ACT AGA GTT CTT TCC TCT hlyA/4285-4310*¹ − TTA ACA TCG AAG C-3′ (SEQID NO: 28) ThromMycBAD 5′-CTG GTT CCG CGT GGA TCT GGG CCC GAApBAD/Myc-HisA/372- + CAA AAA CTC ATC TCA-3′ 395 (SEQ ID NO: 29) HispBAD5′-TCA ATG ATG ATG ATG ATG ATG GTC GAC pBAD/Myc-HisA/414- − GGC-3′ 443(SEQ ID NO: 30) lacZEV-up 5′-CTG CTG CTG CTG AAC GGC AAG-3′ E.coli-lacZ/1021- + (SEQ ID NO: 31) 1041 lacZEV-down 5′-TCA TTG GCA CCATGC CGT GGG-3′ E. coli-lacZ/1250- − (SEQ ID NO: 32) 1270 *¹the numbersrefer to the nucleotide positions of the 8215 bp nucleotide sequence ofthe E. coli plasmid pHly152 encoded hemolysin determinant published byHess et al., 1986, with accession no. M14107. The hemolysin genes hlyC,hlyA, hlyB and hlyD within this sequence are located at the nucleotidepositions 796 to 1308, 1320 to 4394, 4466 to 6589 and 6608 to 8044,respectively.

1. A process for the production of at least one protein of interestcomprising culturing a host cell in a compartment system, which hostcell is stably expressing a secretion system and capable of secretingthe heterologous protein of interest, wherein the protein of interest issecreted by the host cell by means of a secretion signal, and whichcompartment system has at least a first and a second compartment, andwherein the host cell is located in the first compartment, and whereinthe first and second compartment are separated from each other by abarrier, wherein the barrier is permeable for the secreted protein ofinterest, but not permeable for the host cell.
 2. The process accordingto claim 1, wherein the host cell is expressing a secretion systemencoded on a mobile vector.
 3. The process according to claim 2, whereinthe mobile vector is either a plasmid or a cosmid.
 4. The processaccording to claim 1, wherein the barrier is part of the firstcompartment of the compartment system.
 5. The process according to claim1, wherein the barrier is a membrane filter, which membrane filter has apore diameter that prevents host cell migration and allows the diffusionof the secreted protein of interest through the membrane.
 6. The processaccording to claim 1, wherein the first compartment is located above thesecond compartment and the secreted protein of interest will diffuse bymeans of gravity through the barrier from the first compartment to thesecond compartment.
 7. The process according to claim 5, wherein thehost cell is located on the surface of the filter and the secondcompartment is a solid phase attached to the opposite surface of thefilter and which solid phase is capable of retaining the secretedprotein after diffusion through the filter.
 8. The process according toclaim 1, wherein the host cell is either E.coli or Pichia patoris. 9.The process according to claim 1, wherein the secretion of the proteinof interest is effected by growth of the host cell.
 10. The processaccording to claim 5, wherein the first and second compartment are stackmicrowells which are separated by said filter.
 11. The process accordingto claim 1, wherein the protein of interest is obtained in the secondcompartment.
 12. The process according to claim 1, wherein the secretedprotein of interest carries an affinity tag, an epitope tag and/orcarries a protease cleavage site suitable for removal of secretionsignal and/or tag sequences.
 13. The process according to claim 1 forthe production of several different proteins of interest, wherein eachhost cell secreting a respective protein of interest is located in adefined area of the first compartment and each of the secreted proteinsof interest will migrate to a defined area of the second compartment orwherein each host cell secreting a respective protein of interest islocated in different first compartments and each of the secretedproteins of interest will migrate to different second compartments. 14.A process for the production of an array of several different proteinsof interest, comprising culturing host cells in a compartment system,which host cells are stably expressing a secretion system, wherein theproteins of interest are secreted by the host cell by means of secretionsignals, which enables the host cells to secrete the respectiveheterologous protein of interest and which compartment system has atleast a first and a second compartment and wherein the different hostcells are arranged in the form of an array in said first compartment andwherein the first and second compartment are separated from each otherby a barrier, wherein the barrier is permeable for the secreted proteinsof interest, but not permeable for the host cell and wherein thesecreted proteins of interest are received in the second compartment inthe form of an array.
 15. The process according to claim 14, wherein thebarrier is part of the first compartment of the compartment system. 16.The process according to claim 14, wherein the array of proteinscorresponds to the proteome of an organism of interest.